CONFORMATION, STABILITY, AND ACTIVE-SITE CYSTEINE TITRATIONS OF ESCHERICHIA-COLI D26A THIOREDOXIN PROBED BY RAMAN-SPECTROSCOPY

The active-site cysteines (Cys 32 and Cys 35) of Escherichia coli thio redoxin are oxidized to a disulfide bridge when the protein mediates s ubstrate reduction. In reduced thioredoxin, Cys 32 and Cys 35 are char acterized by abnormally low pK(a) values. A conserved side chain, Asp 26, which is sterically accessible to the active site, is also essenti al to oxidoreductase activity. pK(a) values governing cysteine thiol-t hiolate equilibria in the mutant thioredoxin, D26A, have been determin ed by direct Raman spectrophotometric measurement of sulfhydryl ioniza tions. The results indicate that, in D26A thioredoxin, both sulfhydryl s titrate with apparent pK(a) values of 7.5 +/- 0.2, close to values m easured previously for wild-type thioredoxin. Sulfhydryl Raman markers of D26A and wild-type thioredoxin also exhibit similar band shapes, c onsistent with minimal differences in respective cysteine side-chain c onformations and sulfhydryl interactions. The results imply that neith er the Cys 32 nor Cys 35 SH donor is hydrogen bonded directly to Asp 2 6 in the wild-type protein. Additionally the thioredoxin main-chain co nformation is largely conserved with D26A mutation. Conversely, the mu tation perturbs Raman bands diagnostic of tryptophan (Trp 28 and Trp 3 1) orientations and leads to differences in their pH dependencies, imp lying local conformational differences near the active site. We conclu de that, although the carboxyl side chain of Asp 26 neither interacts directly with active-site cysteines nor is responsible for their abnor mally low pK(a) values, the aspartate side chain may play a role in de termining the conformation of the enzyme active site.